Briavioids E–G, Newly Isolated Briarane-Diterpenoids from a Cultured Octocoral Briareum violaceum

The chemical screening of a cultured soft coral, Briareum violaceum, led to the isolation of eight natural, briarane-related diterpenoids, including three unreported metabolites, briavioids E–G (1–3), and five known briaranes, briacavatolides B (4) and C (5), briaexcavatin L (6), briaexcavatolide U (7) and briarenol K (8). The structures of briaranes 1–8 were established using spectroscopic methods. The absolute configuration of briavioid A (9), obtained in a previous study, was reported for the first time in this study by a single-crystal X-ray diffraction analysis using a copper radiation source. The anti-inflammatory activity of briaranes 1 and 2 and briaranes 4–8 was evaluated by screening their inhibitory ability against the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins in lipopolysaccharide (LPS)-induced RAW 264.7 macrophage cells.


Introduction
Every year, thousands of newly isolated marine natural products are reported [1,2]. These metabolites provide a wide range of bioactivities, including anti-cancer, anti-viral, anti-bacterial, anti-fungal and anti-inflammatory abilities [1,2]. These natural products are primarily isolated from marine microorganisms, algae and invertebrates. In this study, we describe the continuing work on the exploration of new substances from cultured marine invertebrates, which may possess interesting bioactivities. Here, eight natural briaranes were obtained from a cultured octocoral, Briareum violaceum (Quoy & Gaimard, 1833) (phylum: Cnidaria, sub-phylum: Anthozoa, class: Octocorallia, order: Scleralcyonacea, family: Briareidae) [3][4][5], including three unreported metabolites, briavioids E-G (1-3), and five reported analogues, briacavatolides B (4) and C (5) [6], briaexcavatin L (6) [7], briaexcavatolide U (7) [8] and briarenol K (8) [9] (Figure 1). Briaranes are a type of 3,8cyclized cembranoid found only in marine invertebrates [10]. Most compounds of this type contain a bicyclo [8.4.0] system and a γ-lactone moiety in their structures and have a potential anti-inflammatory activity [11]. We reported herein the isolation and structural determination of all isolates, as well as the anti-inflammatory profile of compounds 1, 2 and 4-8 using an in vitro assay to screen the reducing ability of these compounds against iNOS and COX-2 protein expression. In addition, the absolute configuration of briavioid A (9) [12] ( Figure 1), obtained in previous study, was further reported for its absolute stereochemistry using a single-crystal X-ray diffraction analysis with a diffractometer equipped with a copper radiation (Cu Kα) source.

Results and Discussion
Mar. Drugs 2023, 21, x FOR PEER REVIEW 4 of 12 19 ( Figure 2), thus establishing the tetracyclic 10/6/5/3 ring system of the briarane scaffold. The HMBC correlations from H3-15/C-1, C-2, C-10, C-14; H3-18/C-8, C-17, C-19 and H3-20/C-10, C-11, C-12 suggested that Me-15, Me-18 and Me-20 are at C-1, C-17 and C-11, respectively. An acetoxymethyl at C-5 was illustrated by the HMBC correlations from H2-16 to C-4, C-5, C-6 and by the long-range allylic couplings between H2-16 and H-6 (J = 1.6, 1.6 Hz) ( Figure 2). HMBC correlations, observed from a hydroxy proton at δH 1.58 (OH-11) to C-10, C-11 and C-20, and the COSY correlation between a hydroxy proton at δH 2.14 (OH-12) and H-12 indicate that these two hydroxy groups are placed at C-11 and C-12, respectively. The H-2 (δH 4.97), H-4 (δH 4.99), H-9 (δH 5.80) and H2-16 (δH 4.76 and 5.25) correlations to the acetate carbonyls at δC 170.5, 169.8, 167.9 and 170.0 confirmed that these four acetoxy groups are positioned at C-2, C-4, C-9 and C-16, respectively. Based on the chemical shifts of oxymethine CH-14 (δC 74.6/δH 4.83, 1H, dd, J = 3.6, 2.0 Hz), the remaining acetoxy group is OAc-14. Fourteen of the fifteen oxygen atoms in the molecular formula of briarane 1 could be accounted for from the presence of one γ-lactone, five esters and two hydroxy groups. The remaining single oxygen atom had to be placed between C-8 and C-17 to form a tetrasubstituted epoxide containing a methyl substituent, based on the 13   The relative stereochemistry of briarane 1 was established by interpreting the NOESY spectrum in addition to the assistance of the computer-generated modeling structure. The literature review indicated that most naturally isolated briaranes have a β-Me and an α-H placed at C-1 and C-10, respectively [10]. From the NOESY data of 1 (Figure 3), H3-15 exhibited cross-peaks with H-14 and one of the diastereotopic methylene protons at C-13 (δH 1.67, H-13β) but not with H-10; while H-10 was correlated to H-2, H-9 and H-12, consistent with the α-orientation of OAc-14 and β-orientations of OAc-2, OAc-9 and OH-12, respectively. Furthermore, H3-20 showed a correlation to H-13β and the absence of cross-peaks with H-10 and H-12, suggesting a β-oriented methyl group at C-11. One of the C-3 diastereotopic methylene protons (δH 2.97) exhibited a correlation to H-7 but not to H-2 and H-4, suggesting the β-orientations of this proton and H-7. The other was assigned as H-3α (δH 2.04). H-4 showed a correlation with H-3α, and a greater coupling constant of 12.4 Hz was noted between H-4 and H-3β, demonstrating the α-orientation of H-4 [13,14] and that the plane between H-4 and H-3β has a dihedral angle of approximately 180°. H-9 correlated to H-10, H3-18 and H3-20, suggesting that H-9 is close to all these protons. In combination with model analysis, Me-18 and 8,17-epoxy group should be placed at β-and α-face in the γ-lactone moiety, respectively. A correlation between H-6 and a proton of the C-16 diastereotopic methylene (δH 5.25) but not with H-7, in addition to a large coupling constant between H-7 and H-6 (J = 8.4 Hz), suggested that the dihedral angle between H-7 and H-6 was nearly 130° [13,14], revealing the Z- The relative stereochemistry of briarane 1 was established by interpreting the NOESY spectrum in addition to the assistance of the computer-generated modeling structure. The literature review indicated that most naturally isolated briaranes have a β-Me and an α-H placed at C-1 and C-10, respectively [10]. From the NOESY data of 1 (Figure 3), H 3 -15 exhibited cross-peaks with H-14 and one of the diastereotopic methylene protons at C-13 (δ H 1.67, H-13β) but not with H-10; while H-10 was correlated to H-2, H-9 and H-12, consistent with the α-orientation of OAc-14 and β-orientations of OAc-2, OAc-9 and OH-12, respectively. Furthermore, H 3 -20 showed a correlation to H-13β and the absence of cross-peaks with H-10 and H-12, suggesting a β-oriented methyl group at C-11. One of the C-3 diastereotopic methylene protons (δ H 2.97) exhibited a correlation to H-7 but not to H-2 and H-4, suggesting the β-orientations of this proton and H-7. The other was assigned as H-3α (δ H 2.04). H-4 showed a correlation with H-3α, and a greater coupling constant of 12.4 Hz was noted between H-4 and H-3β, demonstrating the α-orientation of H-4 [13,14] and that the plane between H-4 and H-3β has a dihedral angle of approximately 180 • . H-9 correlated to H-10, H 3 -18 and H 3 -20, suggesting that H-9 is close to all these protons. In combination with model analysis, Me-18 and 8,17-epoxy group should be placed at βand α-face in the γ-lactone moiety, respectively. A correlation between H-6 and a proton of the C-16 diastereotopic methylene (δ H 5.25) but not with H-7, in addition to a large coupling constant between H-7 and H-6 (J = 8.4 Hz), suggested that the dihedral angle between H-7 and H-6 was nearly 130 • [13,14], revealing the Z-geometry of ∆ 5 . The above interpretation enables the identification the relative configuration of all stereogenic centers of 1 as (1R*,2S*,4R*,7S*,8S*,9S*,10S*,11S*,12S*,14S*,17R*).  Briavioid F (2) was isolated as an amorphous powder. Its HRESIMS peak was at m/z 573.23086, consistent with the molecular formula C28H38O11 (calculated for C28H38O11 + Na, 573.23063) with 10 degrees of unsaturation. The IR spectrum of 2 contained signals of hydroxy (νmax 3293 cm -1 ), γ-lactone (νmax 1785 cm -1 ) and ester (νmax 1735 cm -1 ) functionalities. Analyzing the 1 H, 13 Table 1). The carbon-skeleton of 2, including the positions of the two trisubstituted olefins and the tetrasubstituted epoxide, was fully established by following correlations observed in the COSY and HMBC spectra ( Figure 4). The oxymethine protons H-3 (δH 5.71) and H-4 (δH 6.32) showed HMBC correlations to the acetate carbonyl at δC 168.9 and n-butyrate carbonyl at δC 174.3, confirmed the position of acetoxy and n-butyroxy groups at C-3 and C-4, respectively. H-9 (δH 3.94) correlated to a hydroxy proton resonating at δH 6.45 in the COSY spectrum, suggesting a hydroxy group at C-9. Evaluated on the chemical shifts of H-2 (δH 3.88) and H-14 (δH 4.60), the remaining hydroxy and acetoxy groups should be positioned at C-2 and C-14, respectively. Briavioid F (2) was isolated as an amorphous powder. Its HRESIMS peak was at m/z 573.23086, consistent with the molecular formula C 28 H 38 O 11 (calculated for C 28 H 38 O 11 + Na, 573.23063) with 10 degrees of unsaturation. The IR spectrum of 2 contained signals of hydroxy (ν max 3293 cm -1 ), γ-lactone (ν max 1785 cm -1 ) and ester (ν max 1735 cm -1 ) functionalities. Analyzing the 1 H, 13 Table 1). The carbon-skeleton of 2, including the positions of the two trisubstituted olefins and the tetrasubstituted epoxide, was fully established by following correlations observed in the COSY and HMBC spectra ( Figure 4). The oxymethine protons H-3 (δ H 5.71) and H-4 (δ H 6.32) showed HMBC correlations to the acetate carbonyl at δ C 168.9 and n-butyrate carbonyl at δ C 174.3, confirmed the position of acetoxy and n-butyroxy groups at C-3 and C-4, respectively. H-9 (δ H 3.94) correlated to a hydroxy proton resonating at δ H 6.45 in the COSY spectrum, suggesting a hydroxy group at C-9. Evaluated on the chemical shifts of H-2 (δ H 3.88) and H-14 (δ H 4.60), the remaining hydroxy and acetoxy groups should be positioned at C-2 and C-14, respectively.
In the NOESY data of 2 (   [13,14], and H-2 showed NOE effects with bo H3-15, indicating that the conformation of H-2 is α-oriented. H-4 correlated w not with H-3 and H3-16; with the assistance of the modeling structure, both H should be oriented to β-face. The olefin protons H-6 and H-12 showed correl H3-16 and H3-20, respectively, confirming the Z-geometries of ∆ 5 and ∆ 11 . H-9 to H-10, H3-18 and H3-20 in the NOESY spectrum, suggesting that these proto in space, implying that, in the computer-generated model structure, the Me-1 epoxide should be placed at the β-and α-face of the γ-lactone moiety, r Therefore, based on the above findings, the relative stereochemistry of briavio established as (1R*,2R*,3R*,4R*,7S*,8R*,9S*,10S*,14S*,17R*).   In the NOESY data of 2 ( Figure 5), H3-15 correlated with H-14 and H-10 correlated with H-3 and H-9, illustrating the α-orientation of OAc-14 and β-orientations of Oac-3 and OH-9. H-2 lacks a coupling with H-3, consistent with the dihedral angle of these two protons being approximately 90° [13,14], and H-2 showed NOE effects with both H-14 and H3-15, indicating that the conformation of H-2 is α-oriented. H-4 correlated with H-7 bu not with H-3 and H3-16; with the assistance of the modeling structure, both H-4 and H-7 should be oriented to β-face. The olefin protons H-6 and H-12 showed correlations with H3-16 and H3-20, respectively, confirming the Z-geometries of ∆ 5 and ∆ 11 . H-9 correlated to H-10, H3-18 and H3-20 in the NOESY spectrum, suggesting that these protons are clos in space, implying that, in the computer-generated model structure, the Me-18 and 8,17 epoxide should be placed at the β-and α-face of the γ-lactone moiety, respectively Therefore, based on the above findings, the relative stereochemistry of briavioid F (2) wa established as (1R*,2R*,3R*,4R*,7S*,8R*,9S*,10S*,14S*,17R*). Briavioid G (3) was isolated as an amorphous powder. The molecular formula of 3 was determined as C26H34O11 (10 degrees of unsaturation) based on a positive ion peak a m/z 545.19912, which presented in its HRESIMS spectrum (calculated for C26H34O11 + Na 545.19933). The IR signals of this compound suggested the presences of hydroxy (νmax 3304 cm -1 ), γ-lactone (νmax 1784 cm -1 ) and ester (νmax 1741 cm -1 ) groups. The 1 H and 13 C NMR data of 3 were found to be almost identical to those of 2 (Table 1), except the signals of a Briavioid G (3) was isolated as an amorphous powder. The molecular formula of 3 was determined as C 26 H 34 O 11 (10 degrees of unsaturation) based on a positive ion peak at m/z 545.19912, which presented in its HRESIMS spectrum (calculated for C 26 H 34 O 11 + Na, 545.19933). The IR signals of this compound suggested the presences of hydroxy (ν max 3304 cm -1 ), γ-lactone (ν max 1784 cm -1 ) and ester (ν max 1741 cm -1 ) groups. The 1 H and 13 C NMR data of 3 were found to be almost identical to those of 2 (Table 1), except the signals of a n-butyroxy group in 2 were replaced by the signals for an acetoxy group in 3, indicating that these two compounds different only on the functional group at C-4.
The planar structure of 3, including the positions of OAc-3, OAc-4, OAc-14, OH-2 and OH-9 then was clearly confirmed by analyzing the COSY and HMBC spectroscopic data ( Figure 6). Based on the NMR data of 2 and 3, the stereochemistry of 3 should be similar to the configuration of 2. It then was further confirmed by the combined interpretation of the NOESY correlation and modeling structure (Figure 7). The configuration of 3 was identified as (1R*,2R*,3R*,4R*,7S*,8R*,9S*,10S*,14S*,17R*). and OH-9 then was clearly confirmed by analyzing the COSY and HM data ( Figure 6). Based on the NMR data of 2 and 3, the stereochemistr similar to the configuration of 2. It then was further confirmed b interpretation of the NOESY correlation and modeling structure configuration of 3 was identified as (1R*,2R*,3R*,4R*,7S*,8R*,9S*,10S*,14  Briavioid A (9) was reported in our previous publication [ stereochemistry was established through a combination of the NOESY e single-crystal X-ray diffraction analysis with a molybdenum radiation (M Å) source. However, the configuration obtained from the X-ray analysis w only be considered absolute when the structure contained at least one h in order to determine the absolute configuration of this compound briavioid A (9) obtained in previous publication [12] was recryst diffraction experiment was carried out using a diffractometer equipped 1.54178 Å) radiation source (Flack parameter x = 0.01 (5)). An Oak Ridge T n-butyroxy group in 2 were replaced by the signals for an acetoxy group in 3, indicating that these two compounds different only on the functional group at C-4.
The planar structure of 3, including the positions of OAc-3, OAc-4, OAc-14, OH-2 and OH-9 then was clearly confirmed by analyzing the COSY and HMBC spectroscopi data ( Figure 6). Based on the NMR data of 2 and 3, the stereochemistry of 3 should b similar to the configuration of 2. It then was further confirmed by the combined interpretation of the NOESY correlation and modeling structure (Figure 7). Th configuration of 3 was identified as (1R*,2R*,3R*,4R*,7S*,8R*,9S*,10S*,14S*,17R*).  Briavioid A (9) was reported in our previous publication [12]. Its relativ stereochemistry was established through a combination of the NOESY experiment and a single-crystal X-ray diffraction analysis with a molybdenum radiation (Mo Kα, λ = 0.71073 Å) source. However, the configuration obtained from the X-ray analysis with Mo Kα could only be considered absolute when the structure contained at least one heavy atom. Thus in order to determine the absolute configuration of this compound, the material o briavioid A (9) obtained in previous publication [12] was recrystallized, and th diffraction experiment was carried out using a diffractometer equipped with Cu Kα (λ = 1.54178 Å) radiation source (Flack parameter x = 0.01 (5)). An Oak Ridge Thermal-Ellipsoid Plot (ORTEP) diagram (Figure 8  Briavioid A (9) was reported in our previous publication [12]. Its relative stereochemistry was established through a combination of the NOESY experiment and a single-crystal X-ray diffraction analysis with a molybdenum radiation (Mo Kα, λ = 0.71073 Å) source. However, the configuration obtained from the X-ray analysis with Mo Kα could only be considered absolute when the structure contained at least one heavy atom. Thus, in order to determine the absolute configuration of this compound, the material of briavioid A (9) obtained in previous publication [12] was recrystallized, and the diffraction experiment was carried out using a diffractometer equipped with Cu Kα (λ = 1.54178 Å) radiation source (Flack parameter x = 0.01 (5)). An Oak Ridge Thermal-Ellipsoid Plot (ORTEP) diagram (Figure 8 As briaranes 1-3, in addition to briavioid A (9), were isolated from the same target organism, B. violaceum, it is reasonable to assume on biogenetic grounds that briaranes 1-3 have the same absolute configurations as 9. Therefore, the absolute configurations of 1-3 were suggested to be (1R,2S,4R,7S,8S,9S,10S,11S,12S,14S,17R), (1R,2R,3R,4R,7S,8R,9S, 10S,14S,17R) and (1R,2R,3R,4R,7S,8R,9S,10S,14S,17R), respectively.
The known briaranes 4-8 were identified as briacavatolides B and C, briaexcavatin L, briaexcavatolide U and briarenol K, respectively, based on their spectroscopic data. Including IR, ESIMS, 1 H, 13 C and DEPT NMR data, they were found to be identical with those of the reported data [6][7][8][9].
The anti-inflammation profiles of briaranes 1 and 2 and 4-8 were screened using the in vitro pro-inflammatory assay to test the inhibitory ability of these compounds against the iNOS and COX-2 protein expressions in LPS-induced RAW 264.7 macrophage cells. The results are shown in Table 2. Except for briarane 6 (briaexcavatin L), all isolates exhibited moderate activity to suppress the generation of iNOS but were not active in the inhibition of COX-2. Even briaranes 2 (briavioid F), 7 (briaexcavatolide U) and 8 (briarenol K) exhibited activity to enhance the release of COX-2. It is interesting to note that the most active compound, 2, significantly reduced the release of iNOS to 51.60% at a concentration of 10 μM; however, this compound enhanced the generation of COX-2 to 140.73%. Briaranes 4 (briacavatolide B) and 7 were also found to display a moderate inhibition effect toward iNOS. Briarane 6 did not show activity toward iNOS, implying that the presence of an acetoxy group at C-4 or C-16 would enhance the activity in comparison with the structure and anti-inflammatory activity of 4 and 7. Table 2. Effects of briaranes 1 and 2 and 4-8 (10 μM) on the expression of LPS-induced, proinflammatory iNOS and COX-2 proteins in macrophages.   As briaranes 1-3, in addition to briavioid A (9), were isolated from the same target organism, B. violaceum, it is reasonable to assume on biogenetic grounds that briaranes 1-3 have the same absolute configurations as 9. Therefore, the absolute configurations of The known briaranes 4-8 were identified as briacavatolides B and C, briaexcavatin L, briaexcavatolide U and briarenol K, respectively, based on their spectroscopic data. Including IR, ESIMS, 1 H, 13 C and DEPT NMR data, they were found to be identical with those of the reported data [6][7][8][9].

iNOS COX-2 β-Actin
The anti-inflammation profiles of briaranes 1 and 2 and 4-8 were screened using the in vitro pro-inflammatory assay to test the inhibitory ability of these compounds against the iNOS and COX-2 protein expressions in LPS-induced RAW 264.7 macrophage cells. The results are shown in Table 2. Except for briarane 6 (briaexcavatin L), all isolates exhibited moderate activity to suppress the generation of iNOS but were not active in the inhibition of COX-2. Even briaranes 2 (briavioid F), 7 (briaexcavatolide U) and 8 (briarenol K) exhibited activity to enhance the release of COX-2. It is interesting to note that the most active compound, 2, significantly reduced the release of iNOS to 51.60% at a concentration of 10 µM; however, this compound enhanced the generation of COX-2 to 140.73%. Briaranes 4 (briacavatolide B) and 7 were also found to display a moderate inhibition effect toward iNOS. Briarane 6 did not show activity toward iNOS, implying that the presence of an acetoxy group at C-4 or C-16 would enhance the activity in comparison with the structure and anti-inflammatory activity of 4 and 7.

General Experimental Procedures
The specific rotation values and IR spectra were measured using a JASCO P-2000 digital polarimeter and a THERMO Scientific Nicolet iS5 FT-IR spectrophotometer, respectively. ESIMS and HRESIMS were recorded using a BRUKER 7 Tesla solariX FTMS system. NMR spectra were obtained from a JEOL Resonance ECZ 400S or an ECZ 600R NMR spectrometer, with the residual signals of CHCl 3 (δ H 7.26 ppm) and CDCl 3 (δ C 77.0 ppm) used as the internal standards for 1 H and 13 C NMR, respectively. Coupling constants (J) are provided in Hz. Column chromatography was carried out with a silica gel (230~400 mesh, MERCK) column. Thin-layer chromatography was performed on plates precoated with silica gel 60 F 254 (0.25-mm-thick, MERCK); the plates then sprayed with 10% (v/v) H 2 SO 4 in methanol, followed by heating to visualize the spots. A normal-phase (NP) HPLC was performed using a system comprised of a HITACHI 5110 pump, a RHEODYNE 7725i injection port and a NP column (YMC pack SIL, 5 µm, 12 nm, 250 × 20 mm, YMC group). Reverse-phase (RP) HPLC was performed using a system comprised of a HITACHI L-2130 pump, a HITACHI L-2455 photodiode array detector, a RHEODYNE 7725i injection port and a RP column (Luna 5 µm C18(2) 100 Å, 250 × 21.2 mm, Phenomenex).

Animal Material
The studied organism was cultured by the National Museum of Marine Biology & Aquarium (NMMBA), Taiwan, in an 80 ton culturing tank. Specimens were collected from the tank in December 2016, and the organism was identified through a comparison of the morphology and the micrograph of sclerites with published, scientific descriptions of Briareum violaceum [3][4][5]. A voucher specimen was deposited in the NMMBA (NMMBA-CSC-002).

In Vitro Anti-Inflammatory Assay
The anti-inflammatory activity of briaranes 1 and 2 and 4-8 was tested by evaluating their inhibitory ability against the expression of iNOS and COX-2 pro-inflammatory proteins in LPS-induced RAW 264.7 macrophage cells. The method was described in detail in the previous publications [20,21].

Conclusions
In this study, the chemical composition of a cultured octocoral, identified as Briareum violaceum, was screened, resulting in the isolation of eight natural briaranes, including three new briarines, briavioids E-G (1-3), and five known analogues, briacavatolides B and C, briaexcavatin L, briaexcavatolide U and briarenol K. The structures of all isolated compounds were determined using spectroscopic methods. An in vitro pro-inflammatory assay was also performed to evaluate the ability of briaranes 1 and 2 and 4-8 against the expression of iNOS and COX-2 proteins in LPS-induced RAW 264.7 macrophage cells. The anti-inflammatory activity results are shown in Table 2, and the structure-activity relationships (SAR) among some similar briaranes were also discussed. In addition, the absolute configuration of briavioid A (9) was reported in this study by using a single-crystal X-ray diffraction analysis with a copper radiation source, using the material obtained in previous study [12].